MOLECULAR BASIS AND USE OF A RADIAL GLIA CELL LINE C6-R

放射状胶质细胞在神经发育过程中短暂出现，在细胞迁移和分化中起关键作用。然而，由于它们在培养中不稳定，并且没有具有径向胶质性质的细胞系，因此它们的研究一直很困难。我们通过转染突变的磷酸酶受体，从大鼠C6胶质瘤细胞系中获得了一个稳定的细胞系（C6- r）。C6-R具有放射状形态，在培养和体内刺激神经元迁移。虽然C6- r和C6在培养中都能快速增殖，但C6- r不能像C6那样在大鼠脑内形成肿瘤。由于在许多其他C6克隆中转染的突变受体的表达不能解释C6- r和C6之间的表型差异，因此这种改变很可能是由于在C6- r基因组DNA的Southern印迹中检测到的单个整合位点的遗传破坏。我们假设这种整合事件破坏了促进神经胶质肿瘤形成或抑制神经胶质发生的基因。本提案的目标是了解C6-R的细胞特性和这种独特表型的分子基础。此外，考虑到C6-R细胞在成熟中枢神经系统中沿白质迁移和排列的能力，以及支持神经元迁移的能力，我们将在中枢神经系统中植入C6-R细胞，以探索其在各种情况下促进神经生长的能力，包括损伤后。为了了解C6- r在体内形成肿瘤的能力降低，将其细胞特性，包括增殖和死亡，与亲本C6细胞系进行比较。分子分析将用于确定导致C6转化为C6- r细胞的遗传改变，以及导致其表型差异的表达差异。质粒拯救和基因组克隆方法将用于从质粒插入位点恢复DNA。该位点的基因，以及其他通过C6- r和C6 mRNA的减法抑制杂交（SSH）鉴定的基因将被分析，以确定它们在胶质瘤发生和形成中的作用。这些研究将使我们更好地了解参与神经胶质发育和高级别肿瘤形成的基因。体内C6-R向可形成肿瘤的细胞的逆转研究可能对理解胶质瘤如何发展为高级别恶性肿瘤具有重要意义。最后，放射状胶质细胞系的表征可能会产生促进损伤后神经生长的新方法。

Radial glial cells appear transiently during neural development and play key roles in cell migration and differentiation. However, they have been difficult to study because they are unstable in culture and cell lines with radial glial properties have not been available. We have derived a stable cell line (C6-R) from the rat C6 glioma cell line by transfection with a mutant phosphatase receptor. C6-R has radial morphology and stimulates neuronal migration in culture and in vivo. Although both C6-R and C6 can proliferate rapidly in culture, C6-R is unable to form tumors in rat brain like C6. Since expression of the transfected mutant receptor in many other C6 clones can not account for the phenotypic differences between C6-R and C6, it is likely that the alteration is due to genetic disruption at a single integration site that has been detected in Southern blots of C6-R genomic DNA. We hypothesize that this integration event disrupted genes that either promote glial tumor formation or suppress gliogenesis. The goals of this proposal are to understand the cellular properties of C6-R and the molecular basis of this unique phenotype. In addition, given the ability of C6-R cells to migrate and align along white matter in mature CNS and to support neuronal migration, they will be implanted in the CNS to explore their ability to promote growth of nerves in various situations including following injury. To understand the reduced ability of C6-R to form tumors in vivo, its cellular properties, including proliferation and death, will be compared with the parental C6 cell line. Molecular analysis will be directed to identify the genetic alterations responsible for transformation C6 into C6-R cells and the resulting differences in expression that are responsible for their phenotypic differences. Plasmid rescue and genomic cloning methods will be used to recover DNA from the locus of plasmid insertion. Genes at this locus, and other identified by subtractive suppression hybridization (SSH) using mRNA from C6-R and C6 will be analyzed to determine their roles in gliogenesis and formation of gliomas. These studies will lead to a better understanding of genes that are involved in glial development and in formation of high grade tumors. Studies of the reversion of C6-R to cells that can form tumors in vivo may have implications for understanding how gliomas progress to high grade malignancies. Finally, characterization of radial glial cell lines may yield new methods for promoting nerve growth following injury.